Region-specific changes in gene expression are associated with cognitive deficits in the alpha-synuclein-induced model of Parkinson's disease: A transcriptomic profiling study.

Mild cognitive impairment (MCI) is a common trait of Parkinson's disease (PD), often associated with early motor deficits, eventually evolving to PD with dementia in later disease stages. The neuropathological substrate of MCI is poorly understood, which weakens the development and administration of proper therapies. In an α-synuclein (αSyn)-based model of PD featuring early motor and cognitive impairments, we investigated the transcriptome profile of brain regions involved in PD with cognitive deficits, via a transcriptomic analysis based on RNA sequencing (RNA-seq) technology. Rats infused in the substantia nigra with human α-synuclein oligomers (H-SynOs) developed mild cognitive deficits after three months, as measured by the two-trial recognition test in a Y-maze and the novel object recognition test. RNA-seq analysis showed that 17,436 genes were expressed in the anterior cingulate cortex (ACC) and 17,216 genes in the hippocampus (HC). In the ACC, 51 genes were differentially expressed between vehicle and H-αSynOs treated samples, which showed N= 21 upregulated and N = 30 downregulated genes. In the HC, 104 genes were differentially expressed, the majority of them not overlapping with DEGs in the ACC, with N = 41 upregulated and N = 63 downregulated in H-αSynOs-treated samples. The Gene Ontology (GO) and the Kyoto Encyclopedia of Gene and Genomes (KEGG) analysis, followed by the protein-protein interaction (PPI) network inspection of DEGs, revealed that in the ACC most enriched terms were related with immune functions, specifically with antigen processing/presentation via the major histocompatibility complex (MHC) class II and phagocytosis via CD68, supporting a role for dysregulated immune responses in early PD cognitive dysfunction. Immunofluorescence analysis confirmed the decreased expression of CD68 within microglial cells. In contrast, the most significantly enriched terms in the HC were mainly involved in mitochondrial homeostasis, potassium voltage-gated channel, cytoskeleton and fiber organisation, suggesting that the gene expression in the neuronal population was mostly affected in this region in early disease stages. Altogether results show that H-αSynOs trigger a region-specific dysregulation of gene expression in ACC and HC, providing a pathological substrate for MCI associated with early PD.

miR-365-3p mediates BCL11A and SOX6 erythroid-specific coregulation: A new player in HbF activation.

Hemoglobin switching is a complex biological process not yet fully elucidated. The mechanism regulating the suppression of fetal hemoglobin (HbF) expression is of particular interest because of the positive impact of HbF on the course of diseases such as ß-thalassemia and sickle cell disease, hereditary hemoglobin disorders that affect the health of countless individuals worldwide. Several transcription factors have been implicated in the control of HbF, of which BCL11A has emerged as a major player in HbF silencing. SOX6 has also been implicated in silencing HbF and is critical to the silencing of the mouse embryonic hemoglobins. BCL11A and SOX6 are co-expressed and physically interact in the erythroid compartment during differentiation. In this study, we observe that BCL11A knockout leads to post-transcriptional downregulation of SOX6 through activation of microRNA (miR)-365-3p. Downregulating SOX6 by transient ectopic expression of miR-365-3p or gene editing activates embryonic and fetal ß-like globin gene expression in erythroid cells. The synchronized expression of BCL11A and SOX6 is crucial for hemoglobin switching. In this study, we identified a BCL11A/miR-365-3p/SOX6 evolutionarily conserved pathway, providing insights into the regulation of the embryonic and fetal globin genes suggesting new targets for treating ß-hemoglobinopathies.

Differentiation of single cell derived human mesenchymal stem cells into cells with a neuronal phenotype: RNA and microRNA expression profile.

The adult bone marrow contains a subset of non-haematopoietic cells referred to as bone marrow mesenchymal stem cells (BMSCs). Mesenchymal stem cells (MSCs) have attracted immense research interest in the field of regenerative medicine due to their ability to be cultured for successive passages and multi-lineage differentiation. The molecular mechanisms governing the self-renewal and differentiation of MSCs remain largely unknown. In a previous paper we demonstrated the ability to induce human clonal MSCs to differentiate into cells with a neuronal phenotype (DMSCs). In the present study we evaluated gene expression profiles by Sequential Analysis of Gene Expression (SAGE) and microRNA expression profiles before and after the neuronal differentiation process. Various tissue-specific genes were weakly expressed in MSCs, including those of non-mesodermal origin, suggesting multiple potential tissue-specific differentiation, as well as stemness markers. Expression of OCT4, KLF4 and c-Myc cell reprogramming factors, which are modulated during the differentiation process, was also observed. Many peculiar nervous tissue genes were expressed at a high level in DMSCs, along with genes related to apoptosis. MicroRNA profiling and correlation with mRNA expression profiles allowed us to identify putative important genes and microRNAs involved in the differentiation of MSCs into neuronal-like cells. The profound difference in gene and microRNA expression patterns between MSCs and DMSCs indicates a real functional change during differentiation from MSCs to DMSCs.

The Intranigral Infusion of Human-Alpha Synuclein Oligomers Induces a Cognitive Impairment in Rats Associated with Changes in Neuronal Firing and Neuroinflammation in the Anterior Cingulate Cortex.

Parkinson's disease (PD) is a complex pathology causing a plethora of non-motor symptoms besides classical motor impairments, including cognitive disturbances. Recent studies in the PD human brain have reported microgliosis in limbic and neocortical structures, suggesting a role for neuroinflammation in the development of cognitive decline. Yet, the mechanism underlying the cognitive pathology is under investigated, mainly for the lack of a valid preclinical neuropathological model reproducing the disease's motor and non-motor aspects. Here, we show that the bilateral intracerebral infusion of pre-formed human alpha synuclein oligomers (H-αSynOs) within the substantia nigra pars compacta (SNpc) offers a valid model for studying the cognitive symptoms of PD, which adds to the classical motor aspects previously described in the same model. Indeed, H-αSynOs-infused rats displayed memory deficits in the two-trial recognition task in a Y maze and the novel object recognition (NOR) test performed three months after the oligomer infusion. In the anterior cingulate cortex (ACC) of H-αSynOs-infused rats the in vivo electrophysiological activity was altered and the expression of the neuron-specific immediate early gene (IEG) Npas4 (Neuronal PAS domain protein 4) and the AMPA receptor subunit GluR1 were decreased. The histological analysis of the brain of cognitively impaired rats showed a neuroinflammatory response in cognition-related regions such as the ACC and discrete subareas of the hippocampus, in the absence of any evident neuronal loss, supporting a role of neuroinflammation in cognitive decline. We found an increased GFAP reactivity and the acquisition of a proinflammatory phenotype by microglia, as indicated by the increased levels of microglial Tumor Necrosis Factor alpha (TNF-α) as compared to vehicle-infused rats. Moreover, diffused deposits of phospho-alpha synuclein (p-αSyn) and Lewy neurite-like aggregates were found in the SNpc and striatum, suggesting the spreading of toxic protein within anatomically interconnected areas. Altogether, we present a neuropathological rat model of PD that is relevant for the study of cognitive dysfunction featuring the disease. The intranigral infusion of toxic oligomeric species of alpha-synuclein (α-Syn) induced spreading and neuroinflammation in distant cognition-relevant regions, which may drive the altered neuronal activity underlying cognitive deficits.

Delta-Globin Gene Expression Is Enhanced in vivo by Interferon Type I.

Beta hemoglobinopathies are widely spread monogenic lethal diseases. Delta-globin gene activation has been proposed as a possible approach for curing these pathologies. The therapeutic potential of delta-globin, the non-alpha component of Hemoglobin A2 (α2δ2; HbA2), has been demonstrated in a mouse model of beta thalassemia, while its anti-sickling effect, comparable to that of gamma globin, was established some time ago. Here we show that the delta-globin mRNA level is considerably increased in a Deoxyribonuclease II-alpha knockout mouse model in which type 1 interferon (interferon beta, IFNb) is activated. IFNb activation in the fetal liver improves the delta-globin mRNA level, while the beta-globin mRNA level is significantly reduced. In addition, we show that HbA2 is significantly increased in patients with multiple sclerosis under type 1 interferon treatment. Our results represent a proof of principle that delta-globin expression can be enhanced through the use of molecules. This observation is potentially interesting in view of a pharmacological approach able to increase the HbA2 level.

Deficiency in interferon type 1 receptor improves definitive erythropoiesis in Klf1 null mice.

A key regulatory gene in definitive erythropoiesis is the transcription factor Krüppel-like factor 1 (Klf1). Klf1 null mice die in utero by day 15.5 (E15.5) due to impaired definitive erythropoiesis and severe anemia. Definitive erythropoiesis takes place in erythroblastic islands in mammals. Erythroblastic islands are formed by a central macrophage (Central Macrophage of Erythroblastic Island, CMEI) surrounded by maturating erythroblasts. Interferon-ß (IFN-ß) is activated in the fetal liver's CMEI of Klf1 null mice. The inhibitory effect of IFN-ß on erythropoiesis is known and, therefore, we speculated that IFN-ß could have contributed to the impairment of definitive erythropoiesis in Klf1 knockout (KO) mice fetal liver. To validate this hypothesis, in this work we determined whether the inactivation of type I interferon receptor (Ifnar1) would ameliorate the phenotype of Klf1 KO mice by improving the lethal anemia. Our results show a prolonged survival of Klf1/Ifnar1 double KO embryos, with an improvement of the definitive erythropoiesis and erythroblast enucleation, together with a longer lifespan of CMEI in the fetal liver and also a restoration of the apoptotic program. Our data indicate that the cytotoxic effect of IFN-ß activation in CMEI contribute to the impairment of definitive erythropoiesis associated with Klf1 deprivation.

Reversible disruption of pre-pulse inhibition in hypomorphic-inducible and reversible CB1-/- mice.

Although several genes are implicated in the pathogenesis of schizophrenia, in animal models for such a severe mental illness only some aspects of the pathology can be represented (endophenotypes). Genetically modified mice are currently being used to obtain or characterize such endophenotypes. Since its cloning and characterization CB1 receptor has increasingly become of significant physiological, pharmacological and clinical interest. Recently, its involvement in schizophrenia has been reported. Among the different approaches employed, gene targeting permits to study the multiple roles of the endocannabinoid system using knockout ((-/-)) mice represent a powerful model but with some limitations due to compensation. To overcome such a limitation, we have generated an inducible and reversible tet-off dependent tissue-specific CB1(-/-) mice where the CB1R is re-expressed exclusively in the forebrain at a hypomorphic level due to a mutation (IRh-CB1(-/-)) only in absence of doxycycline (Dox). In such mice, under Dox(+) or vehicle, as well as in wild-type (WT) and CB1(-/-), two endophenotypes motor activity (increased in animal models of schizophrenia) and pre-pulse inhibition (PPI) of startle reflex (disrupted in schizophrenia) were analyzed. Both CB1(-/-) and IRh-CB1(-/-) showed increased motor activity when compared to WT animals. The PPI response, unaltered in WT and CB1(-/-) animals, was on the contrary highly and significantly disrupted only in Dox(+) IRh-CB1(-/-) mice. Such a response was easily reverted after either withdrawal from Dox or haloperidol treatment. This is the first Inducible and Reversible CB1(-/-) mice model to be described in the literature. It is noteworthy that the PPI disruption is not present either in classical full CB1(-/-) mice or following acute administration of rimonabant. Such a hypomorphic model may provide a new tool for additional in vivo and in vitro studies of the physiological and pathological roles of cannabinoid system in schizophrenia and in other psychiatric disorders.